Normalization of polymerase activity
Provided herein is technology relating to the amplification-based detection of nucleic acids and particularly, but not exclusively, to methods and compositions for minimizing variability in the activity between different samples or manufacturing lots of DNA polymerases, such as Taq DNA polymerase.
1. A method, comprising:
a) providing a reaction mixture comprising:
i) a sample suspected of comprising a target nucleic acid;
ii) purified exogenous non-target DNA from fish;
iii) DNA polymerase;
iv) dNTPs;
v) amplification primers configured for amplifying a DNA product from the target nucleic acid;
vi) flap endonuclease; and
vii) an oligonucleotide probe hybridizable to the DNA product;
wherein the reaction mixture is characterized in that it can amplify the DNA product from the target nucleic acid if the target nucleic acid is present in the sample; and
b) conducting an amplification reaction with the reaction mixture wherein, if the target nucleic acid is present in the reaction mixture, an oligonucleotide probe hybridized to the DNA product is cleaved by the flap endonuclease.
2. The method of claim 1 , wherein the target nucleic acid comprises DNA and/or RNA.
3. The method of claim 2 , wherein the target nucleic acid is synthetic.
4. The method of claim 2 , wherein the target nucleic acid comprises plasmid DNA.
5. The method of claim 1 , wherein the target nucleic acid comprises nucleic acid from a human subject.
6. The method of claim 5 , wherein the nucleic acid from a human subject comprises genomic DNA.
7. The method of claim 1 , wherein the oligonucleotide probe is a flap oligonucleotide.
8. The method of claim 1 , wherein the reaction mixture comprises an oligonucleotide with a fluorescent label.
9. The method of claim 8 , wherein the oligonucleotide with a fluorescent label is a probe oligonucleotide hybridizable to the DNA product.
10. The method of claim 1 , wherein the flap endonuclease is a FEN-1 endonuclease.
11. A composition, comprising:
i) a sample suspected of comprising a target nucleic acid;
ii) purified exogenous non-target DNA from fish;
iii) DNA polymerase;
iv) dNTPs;
v) amplification primers configured for amplifying a DNA product from the target nucleic acid;
vi) flap endonuclease; and
vii) an oligonucleotide probe hybridizable to the DNA product.
12. The composition of claim 11 , wherein the target nucleic acid comprises DNA and/or RNA.
13. The composition of claim 12 , wherein the target nucleic acid is synthetic.
14. The composition of claim 12 , wherein the target nucleic acid comprises plasmid DNA.
15. The composition of claim 11 , wherein the target nucleic acid comprises nucleic acid from a human subject.
16. The composition of claim 15 , wherein the nucleic acid from a human subject comprises genomic DNA.
17. The composition of claim 11 , wherein the oligonucleotide probe is a flap oligonucleotide.
18. The composition of claim 11 , wherein the composition comprises an oligonucleotide with a fluorescent label.
19. The composition of claim 18 , wherein the oligonucleotide with a fluorescent label is a probe oligonucleotide hybridizable to the DNA product.
20. The composition of claim 11 , wherein the flap endonuclease is a FEN-1 endonuclease.